U.S. patent number 6,813,568 [Application Number 10/203,235] was granted by the patent office on 2004-11-02 for system and process for microfluidics-based automated chemistry.
This patent grant is currently assigned to Memorial Sloan-Kettering Cancer Center. Invention is credited to Michael Powell, Paul Tempst.
United States Patent |
6,813,568 |
Powell , et al. |
November 2, 2004 |
System and process for microfluidics-based automated chemistry
Abstract
A system for carrying out reactions that includes a small volume
rotary selector valve having a plurality of peripheral ports and a
small volume rotary switching valve, also having a plurality of
peripheral ports, where the selector valve and switching valve are
controlled by a computer. The rotary selector valve is connected by
common port to a peripheral port of the rotary switching valve. The
internal volumes of the rotary selector valve and rotary switching
valves are 1.5 .mu.l or less.
Inventors: |
Powell; Michael (Boston,
MA), Tempst; Paul (New York, NY) |
Assignee: |
Memorial Sloan-Kettering Cancer
Center (New York, NY)
|
Family
ID: |
27734006 |
Appl.
No.: |
10/203,235 |
Filed: |
December 11, 2002 |
PCT
Filed: |
January 09, 2002 |
PCT No.: |
PCT/US02/00370 |
PCT
Pub. No.: |
WO02/05518 |
PCT
Pub. Date: |
July 18, 2002 |
Current U.S.
Class: |
702/31; 422/521;
435/286.5; 73/864.84 |
Current CPC
Class: |
B01J
19/0046 (20130101); B01L 3/502738 (20130101); B01L
3/567 (20130101); F16K 11/0743 (20130101); G01N
35/00 (20130101); G01N 35/1097 (20130101); B01L
2200/027 (20130101); B01L 2400/0622 (20130101); G01N
2030/8435 (20130101); G01N 30/82 (20130101); G01N
2030/027 (20130101); B01L 2400/0644 (20130101) |
Current International
Class: |
B01L
3/00 (20060101); B01J 19/00 (20060101); F16K
11/074 (20060101); F16K 11/06 (20060101); G01N
1/00 (20060101); G01N 35/00 (20060101); G01N
30/02 (20060101); G01N 30/00 (20060101); G01N
30/82 (20060101); G01N 30/84 (20060101); G06F
019/00 (); G01N 031/00 (); G01N 001/10 () |
Field of
Search: |
;702/31
;73/864.83,864.84,61.52 ;418/63 ;422/63,64,65,67,99,100
;435/6,91.2,287.1,286.5,289.1 ;356/73 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Barlow; John
Assistant Examiner: Le; John
Attorney, Agent or Firm: Klauber & Jackson
Government Interests
The research leading to the present invention was supported, at
least in part, by NSF grant DBI-942012 and Development Funds from
NCI Grant P30 CA08748. Accordingly, the Government may have certain
rights in the invention.
Parent Case Text
This application claims priority from PCT/US02/00370 field on Jan.
9, 2002.
Claims
What is claimed is:
1. A system for carrying out one or more chemical reactions, said
system comprising a rotary selector valve and a rotary switching
valve, each valve under the control of a computer, the internal
volumes of the selector and switching valves each being 1.5 .mu.l
or less wherein the rotary selector valve comprises, in a first
unit, a central common port and a circular array of a plurality of
peripheral ports such that the main axis of the common port is the
same as is the main axis through the circular array, and wherein
said valve comprises, on a second unit, a radial connector channel,
the first and second units being juxtaposed such that there is a
single continuous selector channel formed by the common port, the
connector channel, and a peripheral port, the selection of the
peripheral port under control of the computer, the internal volume
of the continuous selector channel being the internal volume of the
valve, and wherein the rotary switching valve comprises a circular
array of three or more peripheral ports in a first unit and a
connector channel in a second unit, the two units being juxtaposed
such that there is a continuous switching channel formed by a first
peripheral port, the connector channel, and a second peripheral
port, the selection of two peripheral ports being under the control
of the computer, the internal volume of the continuous switching
channel being the internal volume of the valve.
2. A system of claim 1 wherein the rotary selector valve is
connected by its central common port to a first peripheral port of
the rotary switching valve, the connection being accomplished by a
conduit means having an internal volume between 0.5 .mu.L and 10
.mu.L.
3. A system of claim 2 wherein the total internal volume of the
selector and switching valves are each in the range 0.04 .mu.L to
1.5 .mu.L.
4. A system of claim 3 wherein the total internal volume of the
selector and switching valves are in the range 0.1 .mu.L to 0.5
.mu.L.
5. A system of claim 2, said system further comprising: a reaction
vessel connected to the second peripheral port in the continuous
switching channel of the switching valve, a first and second
reagent vessel, said vessels each connected to first and second
peripheral ports of the selector valve, said vessels each connected
to a gas source comprising a gas under pressure.
6. A system of claim 5, the selector valve thereof being a first
selector valve and the switching valve thereof being a first
switching valve, said system further comprising: a second rotary
selector valve connected to a second rotary switching valve; a
third rotary switching valve; a conversion vessel connected to
receive output from said third rotary switching valve, a
restraining means for restraining unprocessed polymer in the
reaction vessel; third and fourth reagent vessels connected to
provide fluid to said conversion vessel; wherein said second rotary
valve, second switching valve, and third switching valve are each
under the control of the computer and each have an internal volume
of 1.5 .mu.l or less.
7. A reaction process under the control of a computer said process
comprising the steps of: 1) Delivering, from a reagent vessel under
pressure from a gas source, a volume of a reagent via a delivery
line to a reaction vessel wherein the total volume delivered for
purposes of a reaction in the reaction vessel is in the range 0.5
uL to 10 uL, and wherein flow through the delivery line is under
the control of a computer-controlled rotary switching valve
connected to a rotary selector valve; 2) Washing said valves and
delivery line with a wash-solvent: 3) Repeating step (1) optionally
with a reagent, reagent vessel and/or gas source different from
that used in step (1) (4) Placing a polymer in a reaction vessel,
said polymer restrained in said vessel; (5) Delivering to said
reaction vessel, from a first reagent vessel under pressure from a
gas source, a volume of a first fluid, said fluid comprising a
first reagent, which reagent will bind to a terminal monomer on the
polymer; (6) Delivering to said reaction vessel, from a second
reagent vessel under pressure from a gas source, a volume of a
second fluid, which fluid will cause a terminal monomer-reagent
moiety to be cleaved from the polymer; (7) causing all or part of
the fluid in the reaction flask to be transferred under gas
pressure via a delivery line to a conversion flask while allowing
the polymer, less the terminal monomer, to remain in the reaction
vessel; (8) Prior or after step (7), delivering from a third
reagent vessel under pressure from a gas source a volume of a third
fluid such that the combination of said fluid and the fluid
transferred in step (7) to the reaction flask will cause the
terminal monomer-reagent moiety to be cleaved to create a terminal
monomer free of covalently bound reagent; (9) Transferring under
gas pressure from a gas source said terminal monomer created in
step (8) to an analytical device that will identify the nature of
the terminal monomer; wherein each volume delivered in steps (5),
(6), (7), (8), is in the range 0.2 uL to 10 uL, and the volume
delivered in step (9) is in the range 1 uL to 10 uL.
8. A process of claim 7 wherein steps (4)-(9) are performed a
plurality of times.
9. A process of claim 7, wherein the process comprises sequentially
delivering acid, organic solvent, and base to the reaction
vessel.
10. A process of claim 7 wherein the reaction vessel is kept free
of air and oxygen.
11. A process of claim 7 wherein the reaction vessel and the
conversion vessel are kept free of air and oxygen.
Description
BACKGROUND
This invention relates to the field of microfluidics-based
automated chemistry.
Many analytical chemical problems involve the analysis of small
amounts of a sample. The ability to do chemical reactions in small
volumes of liquid is important in such cases as the reaction rate
can decrease to commercially unacceptable levels if the
concentration of the sample is too low. Additionally, for
analytical devices used to monitor the results of such reactions,
it is frequently important that the reaction product be in a small
volume in order that a detectable concentration be present. The
present invention offers a solution to the problem of doing
chemical reactions in small volumes, to doing a succession of such
reactions, and to doing them on an automated basis.
Although the present invention will be seen to have general
chemical applicability, its application to biomedical research is
of particular interest. Post-genome research will likely focus on
understanding the cellular chemistry, circuitry and communications
underlying life's vital processes. Biomedical scientists, for
instance, will aim to identify how genetic determinants of disease
alter cellular physiology and response to agonists. Predictably,
all this will involve biochemical analysis of larger numbers of
samples containing ever lower concentrations of analyte. In most
cases, analyses entail multistep procedures, including chemical and
enzymatic reactions. Not only will automation prove essential in
such cases, it must also be done in fully integrated instruments
that incorporate the smallest possible reaction vessels and wetted
surfaces. Progress towards this goal has come from nano-fabricated
devices.sup.1-4 ("lab-on-a-chip"), but severe limitations on sample
volume may restrict the technology to fast, parallel analysis of
abundant and/or amplifiable (e.g. by PCR) molecules. What's needed
is automation whereby trace analytes are processed in their
entirety. We wanted to construct such a device, initially using
chemical protein sequencing as a model system. Such chemistries
have been replaced during recent years by mass spectrometry as a
means to protein identification..sup.5-8 Yet, the fact remains that
chemical analysis can yield rather long stretches of easy to
interpret sequence, including on intact proteins, with the caveat
that current instruments are at least ten to twenty times less
sensitive than most mass analyzers.
Traditionally, protein chemical sequencing is done by stepwise
removal of amino acids from the N-teminal end, one at a time. In
this method, the Edman degradation,.sup.9,10 phenyl isotliocyanate
(PITC) is coupled to the alpha-amino group of the polypeptide to
form a phenyl thiocarbamyl (PTC)-derivative; anhydrous acid causes
selective release of the PTC-amino, leaving a truncated peptide
chain. The resulting anilino thiazolinone amino acid (ATZ-aa) is
converted with aqueous acid to a more stable phenyl thiohydantoin
amino acid (PTH-aa) and identified. The latter step cannot be done
in the presence of polypeptide as it would cause hydrolysis; thus,
the ATZ-aa must be extracted. The procedure was partially
automated, first by Edman in the `spinning cup` version,.sup.11 and
later in Laursen's solid-phase sequencer..sup.12 Conversion was
initially done outside the machine, but later incorporated into the
automated process by Wittman-Liebold..sup.13 Until then, thin layer
or gas chromatography were used for PTH-aa identification;
Hunkapiller and Hood were among the first to routinely use
reversed-phase high performance liquid chromatography (RP-HPLC) for
this purpose..sup.14 Around 1980, the gas-phase (GP) sequencer was
developed by Hewick and coworkers..sup.15 Here, wash-out of the
peptide from the reaction vessel was prevented by non-covalent
immobilization on a glass-fibre disc and by delivering polar
liquids as vapors. The process has been further automated by
coupling HPLC identification of PTH-aa's `on-line` with the
sequencer; contents of the conversion flask are hereby directly
transferred into the LC injector loop. Since then, progress has
come from incremental improvements such as femtomole level HPLC
detection,.sup.16 rigorous instrument optimization and maintenance
routines,.sup.17 and the use of a smaller reaction
cartridges..sup.17-19 Combined with improved micro-preparation of
polypeptides,.sup.20-22 chemical sequencing at the 1-3 picomole
level is currently possible and extended sequencing runs with
femtomole level signal have been reported..sup.16,23,24 Additional
modifications to the process have since been suggested that should
allow true femtomole level sequencing. All relate to increased
sensitivities of amino acid derivative detection. This can be
accomplished by miniaturizing the HPLC-based PTH-aa detection
system, or by producing modified Edman end-products of higher
detect ability, or both.
Whereas femtomole level PTH-aa separations on microbore columns can
be done,.sup.19,25 the use of capillary columns (300-micron ID) is
more problematic, as injection volumes of over 0.5 .mu.L cause
baseline disturbances..sup.26 Complete injection (or even 10%) of
the sample in a 0.5-.mu.L volume is not possible from any
commercial automated sequencer. This also applies to capillary zone
electrophoresis (CZE)-based amino acid-derivative detection
systems..sup.27
Considerable effort has been expended to generate fluorescent amino
acid derivatives as end-products of Edman chemistry. Fluorescent
sequencing is especially appealing since the introduction of
sub-attomole amino acid analysis by CZE with laser-induced
fluorescence..sup.27,28 Yet, again, loading volumes are in the
nanoliter range, several orders of magnitude less than what is
typically present in a sequencer flask. Another approach to
`high-detect ability` is the generation of quaternary or tertiary
amine thiohydantoin amino acid derivatives, analyzable by mass
spectrometry at the low femtomole level..sup.29,30 However, both
methods never progressed beyond the R&D stage.
Development of any Edman based, femtomole-level technique requires
satisfying two major criteria: (i) quantitative transfer, in the
smallest possible volume, of amino acid derivatives to the site of
analysis; and (ii) reducing chemical background that will impede
any ultra-sensitive analytical technique. This can only be
accomplished by further miniaturization of the chemistry.
We describe a microfluidics-based instrument, consisting of
multiple rotary valves, capillary tubing and miniaturized reaction
vessels, for the purpose of performing automated chemical and
biochemical reactions on a very small scale. Close to 100% of the
reaction end-products are available in a minimal volume (.ltoreq.5
.mu.L) inside a pressurized mirco-vial for subsequent analysis.
This makes the system compatible with capillary HPLC and, in
principle, with continuous flow nano-electrospray mass
spectrometry. Total control of flow path combinations and
directions, temperatures and gas pressures enables precise
execution of complex biochemical laboratory procedures. Instrument
performance was convincingly demonstrated by partially sequencing
100 femtomoles of an intact protein using classical Edman chemistry
in combination with capillary-bore liquid chromatography. To our
knowledge, this is the smallest amount of protein ever reported to
be successfully analyzed in this way.
SUMMARY OF THE INVENTION
The invention is a chemical system and related processes that
utilize small-volume rotary selector valves and small-volume rotary
switching valves in combination under the control of a computer.
The small rotary valves are particular well-suited to computer
control. As a result, a single system can be programmed to do a
variety of tasks by changing the program but leaving the system's
components substantially intact.
As a result, in a general aspect the invention is a system for
carrying out one or more chemical reactions, said system comprising
a rotary selector valve and a rotary switching valve, each valve
under the control of a computer, the internal volumes of the
selector and switching valves each being 1.5 .mu.l or less.
Specific aspects of the invention that are of particular interest
are the selector valve-switching valve combination, a core system
for regulating a sequence of reactions, a core system adapted for
polymer sequence analysis, and processes that implement the
systems. The core systems can, as indicated by the use of the word
"comprising" in their description, be either used as stand-alone
systems or be part of larger systems.
Selector Valve-Switching Valve Combination
An important aspect of the invention is a rotary valve combination
comprising a rotary selector valve (with its plurality of
peripheral ports) connected by its central common port to a
peripheral port of a rotary switching valve. The connection is
preferably accomplished by a conduit means (e.g., a tube or
capillary). It is preferred that the conduit means have an internal
volume between 0.5 .mu.L and 10 .mu.L.
The Rotary Selector Valves
It is preferred that the rotary selector valve comprise, in a first
unit (e.g., the stator), a central common port and a circular array
of a plurality of peripheral ports such that the main axis of the
common port is the same is as the main axis through the circular
array, and wherein said valve comprises, on a second unit, a radial
connector channel, the first and second units being juxtaposed such
that there is a single continuous selector channel formed by the
common port, the connector channel, and a peripheral port, the
selection of the peripheral port under control of the computer (the
control preferentially effectuated by rotating the second unit),
the internal volume of the continuous selector channel being the
internal volume of the valve,
Preferably the two units are positioned in flat contact with each
other, the flat contacting surface of the first unit against the
flat contacting surface of the second unit. Contact between the two
units is such that both the common port and a peripheral port meet
the connector channel. This is optimally done by constructing the
connector channel as a groove on the contacting surface of the
second unit. It is preferred, but not required, that the common
port act as an output port and that the connected peripheral port
act as an input port, but the reverse relationship between the
ports is also possible. The total internal volume (central port
plus connector channel plus peripheral port) is preferably 1.5
.mu.L or less (more preferably in the range 0.04 .mu.L to 1.5
.mu.L, most preferably 0.1 .mu.L to 0.5 .mu.L).
The Rotary Switching Valves
It is preferred that the rotary switching valve comprise a circular
array of three or more peripheral ports in a first unit (e.g., the
stator) and a connector channel in a second unit, the two units
being juxtaposed such that there is a continuous switching channel
formed by a first peripheral port, the connector channel, and a
second peripheral port, the selection of two peripheral ports being
under the control of the computer (the control preferentially
effectuated by rotating the second unit), the internal volume of
the continuous switching channel being the internal volume of the
valve.
Preferably the two units are positioned in flat contact with each
other, the flat contacting surface of the first unit against the
flat contacting surface of the second unit. Contact between the two
units is such that two peripheral ports meet the connector channel.
This is optimally done by constructing the connector channel as a
groove on the contacting surface of the second unit. (The groove
can be straight or curved). The total internal volume (central port
plus connector channel plus peripheral port) is preferably 1.5
.mu.L or less (more preferably, in the range 0.04 .mu.L to 1.5
.mu.L, most preferably 0.1 .mu.L to 0.5 .mu.L).
System For Regulating a Sequence of Reactions
In a general aspect the invention is a computerized system for
regulating complex reaction sequences in small volumes of solution,
said system comprising: a reaction vessel (for holding a volume of
fluid in which a reaction takes place); a first and second reagent
vessel (for holding fluid reagents), said vessels each connected to
a gas source, a rotary valve combination connected to receive fluid
from said reagent vessels and to deliver fluid to said reaction
vessel, said combination optionally with other receiving-delivery
capabilities, said rotary valve combination comprising a
multi-position rotary selector valve connected to a rotary
switching valve; a computer connected to said valve combination so
that said computer controls both whether the switching valve is
configured to allow or to not allow fluid flow to the reaction
vessel and whether the selector valve is configured for input from
the first or second reagent vessel; and wherein the internal volume
of each of said valves along the path of fluid flow, is 1.5 .mu.L
or less, (preferably in the range 0.04 .mu.L to 1.5 .mu.L, more
preferably 0.1 .mu.L to 0.5 .mu.L).
Each gas source is selected from a group of one or more gas sources
each comprising a gas under pressure, said gas optionally differing
from source to source. Any gas or gas combination that is not
chemically reactive with a fluid or other reagent in the system can
be used.
System Adapted for Polymer Sequence Analysis
In another aspect, the selector valve of the above-noted system is
a first selector valve and the switching valve thereof is a first
switching valve, and the system further comprises: a second rotary
selector valve connected to a second rotary switching valve; a
third rotary switching valve; a conversion vessel connected to
receive output from said third rotary switching valve, a
restraining means for restraining unprocessed polymer in the
reaction vessel; third and fourth reagent vessels connected to
provide fluid to said conversion vessel; wherein said second rotary
valve, second switching valve, and third switching valve are each
under the control of the computer and each have an internal volume
of 1.5 .mu.l or less (preferably 0.04 .mu.L to 1.5 .mu.L, more
preferably 0.1 .mu.L to 0.5 .mu.L).
In particular embodiments, the system further comprises one or more
of the following: one or more wash solution vessels (each for
holding a volume of fluid, preferably a wash solution), each said
vessel connected to a gas source, each said vessel providing
optional input (directly or via other elements of the system) via a
selector valve to the reaction vessel and/or conversion vessel; and
a switching valve, under the control of the computer, for
controlling fluid flow to an analytical device (such as an HPLC
column or electrospray ionization mass spectrometer) from the
conversion flask.
Reaction Vessel and Conversion Vessel
It is preferred that, when the first and second fluid reagents are
in the reaction vessel, the volume of fluid in the reaction vessel
is in the range of 0.5 .mu.L to 10 .mu.L (preferably 1 .mu.L to 5
.mu.L).
It is preferred that a volume of fluid in the conversion vessel be
in the range 1 .mu.L to 150 .mu.L (preferably 3 .mu.L to 50 .mu.L,
most preferably 3 .mu.L to 15 .mu.L).
Selector Valve Cascade
An aspect of the invention is a rotary selector valve cascade in
which a plurality of peripheral ports of a first selector valve are
each connected to the central common port of one of plurality of
rotary selector valves that each comprise a plurality of peripheral
ports, the total internal volume of each valve being 1.5 .mu.L or
less (more preferably in the range of 0.4 .mu.L to 1.5 .mu.L, most
preferably 0.1 .mu.L to 0.5 .mu.L).
Process Aspect of the Invention
In another general aspect, the invention is a reaction process
under the control of a computer said process comprising the steps
of: 1) Delivering, from a reagent vessel under pressure from a gas
source, a volume of a reagent via a delivery line to a reaction
vessel wherein the total volume delivered for purposes of a
reaction in the reaction vessel is in the range 0.5 .mu.L to 10
.mu.L, preferably 1 .mu.L to 5 .mu.L, and wherein flow through the
delivery line is under the control of a computer-controlled rotary
valve combination; Preferred is the process with an additional
steps (2) and (3): 2) Washing said valve combination and delivery
line with a wash-solvent; and 3) Repeating step (1) optionally with
a reagent, reagent vessel and/or gas source different from that
used in step (1).
Process Aspect of the Invention Used to Sequence Polymers
In an aspect of the invention adapted for sequencing a polymer, the
process comprises the steps of: (1) Placing a polymer (preferably
in the range 5 to 500 femtomoles, more preferably 20 to 250
femtomoles, most preferably 50 to 200 femtomoles) in a reaction
vessel, said polymer restrained in said vessel, the restraining
preferably accomplished by adsorption of the polymer to a solid
support; (2) Delivering to said reaction vessel, from a first
reagent vessel under pressure from a gas source, a volume of a
first fluid, said fluid comprising a first reagent, which reagent
will react with a terminal monomer on the polymer; (3) Delivering
to said reaction vessel, from a second reagent vessel under
pressure from a gas source, a volume of a second fluid, which fluid
(the fluid can be a solvent or a solution of a solute in a solvent)
will cause a terminal monomer-reagent moiety to be cleaved from the
polymer; (4) causing all or part of the fluid in the reaction flask
to be transferred under gas pressure via a delivery line to a
conversion flask while allowing the polymer, less the terminal
monomer, to remain in the reaction vessel; (5) Prior or after step
(4), delivering from a third reagent vessel under pressure from a
gas source a volume of a third fluid such that the combination of
said fluid and the fluid transferred in step (4) to the reaction
flask will cause the terminal monomer-reagent moiety to be cleaved
to create a terminal monomer free of covalently bound reagent; (6)
Transferring under gas pressure from a gas source said terminal
monomer created in step (5) to an analytical device that will
identify the nature of the terminal monomer.
It is preferred that each volume delivered in steps (2), (3), (4),
(5), is in the range 0.2 .mu.L to 10 .mu.L, (preferably 0.5 .mu.L
to 5 .mu.L). Independently, it is preferred that the volume
delivered in step (6) is in the range 1 .mu.L to 10 .mu.L,
(preferably 2 .mu.L to 5 .mu.L).
In a preferred embodiment of the process adapted for sequencing a
polymer, the process comprising steps (1)-(6), is performed a
plurality of times.
Air-Free Reaction System
In preferred embodiments of the processes, the reaction vessel and
the conversion vessel are kept free of air and oxygen. The ability
to do this effectively is an advantage of the present
invention.
Sequential Treatment with Acid and Base
In particular embodiments of the processes, the process comprises
sequentially delivering acid, organic solvent, and base to the
reaction vessel. The ability to do this effectively is an advantage
of the present invention.
Applications of the Invention
Examples of such applications are: 1) automated reactor for
solution chemistry; 2) automated reactor for liquid "flow-through"
chemistry; 3) automated reactor for gas-phase chemistry; 4)
automated sample preparation, modification, and reaction prior to
electrospray ionization mass spectrometry; 5) automated enzymatic
digestion of proteins, nucleic acids and complex carbohydrates; 6)
thermostatic control for sample preparation, modification and
reaction (including the above uses); 7) automatic precision volume
metering utilizing loops with volume range 0.1 microliter and
above; 8) automated selection of samples for any use described
above.
Specific Polymer Applications
In one set of embodiments, the system is adopted for processing a
polymer in step with fashion, one monomer at a time.
Example of such processing are: 1) the determination of the amino
acid sequence of a polypeptide; 2) the determination of the base
sequence of a nucleic acid; and 3) the determination of the sugar
sequence of a polysaccharide;
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1.(A) Schematic diagram of the `PSMSK` automated microfluidics
system. The instrument 100 is shown here in a protein sequencer
configuration. Connections of the computer 17 to the other
components of the system are not shown but are described in the
text. (B) Legend to selected symbols seen in (A). (C) Examples of
materials used with system shown in (A).
FIG. 2. PSMSK `function #29`, deliver liquid TFA to cartridge. This
function consists of the seven sequential `events`, i.e. unique
valve position combinations, as shown in panels A-G and listed in
panel H where events 1-7 correspond to panels A-G, respectively.
Only rotary valves 51, 61, 66 are shown; for the precise
positioning in the entire system and for the key to symbols and
abbreviations, see schematic in FIG. 1. Valve 51 is a ten-port
selection and valves 61, 66 are two-way switching (A/B); solenoid
valves (not shown) are used to pressurize or vent the TFA bottle
(ON/OFF). The flow path through the valves during each event is
shown in bold; no liquid or gas flow occurs during `event #7`.
Total function time is 1 min 44 sec. `Function #29` is used in the
reaction cycle (Table 2A) as step 30.
FIG. 3. `On-line` capillary-LC analysis of 25 fmol (delivered to
the flask) of PTH-amino acid standard under conditions as described
in the text. A 300-micron i.d. column, packed with C.sub.18 PTH
(5-micron particle size) resin, was used at a flow of 3 .mu.L/min;
absorbance detection was done at 269 nm. Only the relevant portion
of the chromatogram is shown. More details can be found in the
`Materials and Methods` section.
FIG. 4. Micro-chemical sequencing of 100 fmol bovine
.beta.-lactoglobulin using the `PSMSK` microfluidics system and
on-line, capillary-LC PTH-amino acid analysis. Experimental
conditions were as listed under `Materials and Methods` and in the
FIG. 3 legend. Chromatograms 1-16 are shown; absorbance detection
full scale varies, as indicated. PTH-amino acid peaks are indicated
with an arrow and the femtomole amounts (corrected for background)
shown.
FIG. 5. Schematic drawing of selected portions of a selector valve.
A. A first side planar view. B. A sectional view. C. A second side
planar view.
FIG. 6. Schematic drawing of selected portions of a switching
valve. A. A first side planar view. B. A sectional view. C. A
second side planar view.
FIG. 7. Top Block of reaction vessel. A. A planar view. B. A
sectional drawing along the line B'--B' in A. C. A bottom planar
view.
FIG. 8. Bottom block of reaction vessel. A. A bottom planar view.
B. A sectional drawing along the line B'--B' in A. C. A top planar
view.
FIG. 9. Schematic sectional view of reaction vessel chamber and
contents created by combining the bottom block (FIG. 8) and top
block (FIG. 7) of the reaction vessel with additional
components.
FIG. 10. Schematic partially sectional drawing of selected portions
of a conversion flask.
FIG. 11. Schematic partially sectional view drawing of a selected
portion of a rotary valve combination in which a valve (FIG. 6) is
connected via a capillary to a selector valve (FIG. 5).
DETAILED DESCRIPTION OF THE INVENTION
Abbreviations and Glossary
ATZ-aa stands for anilino thiazolinone amino acid.
BLG stands for beta-lactoglobulin.
BuCl stands for butyl chloride.
CLC stands for capillary liquid chromatography
A computer is an electronic device that receives input information
in electronic for and process that information electonically or
electromagnetically to create electronic output signals that can be
converted to printed material, converted electronically displayed
material, or otherwise further processed. Typical computers are
personal computers such as made by IBM or other comapnies but both
more sophisticated computers can also be used and, conversely, the
computer can be one of much simpler construction than a personal
computer by virtue of the fact that it is dedicated to the process
and systems of the invention.
CZE stands for capillary zone electrophoresis.
DMPTU stands for dimethylphenylthiourea.
DPTU stands for diphenylthiourea
EtAc stands for ethyl acetate.
GP stands for gas-phase.
HPLC stands for high performance liquid chromatography.
LC stands for liquid chromatography.
Lys stands for lysine.
10% MeCN and 10% MeCN/H.sub.2 O stands for 10% acetonitrile in
water.
MS/MS stands for tandem mass spectrometry.
ESI stands for electrospray ionization.
PCR stands for polymeric chain reaction.
PITC stands for phenyl isothiocyanate.
Plurality means 2 or more.
PSMSK stands for the automated micro-fluidics system 100.
PTC stands for phenyl thiocarbamyl.
PTH-aa stands for phenyl thiohydantoin amino acid.
Reagents include any fluids or solids that participate in chemical
reactions and also any solution or solution components in which
said fluids or solids are dissolved or suspended.
RP-HPLC stands for reversed-phase high performance liquid
chromatography
25% TFA and 25% TFA/H.sub.2 O stands for 25% TFA (v/v) in H.sub.2
O
0.1% TFA stands for 0.1% TFA in H.sub.2 O
TFA stands for trifluoroacetic acid.
THF stands for tetrahydrofuran.
TMA stands for trimethylamine.
The terms "path of fluid flow" and "flow through channel" are used
interchangeably. For a particular component of the system in a
given configuration (e.g., a particular valve setting), the terms
reprsent the total path followed by fluid within that component in
that configuration.
"Under pressure" means under greater than atmospheric pressure.
Val stands for valine.
A "volumetric loop" is a conduit that can be either straight,
looped, helical, or otherwise curved. "Loop" here is a term of
art.
"Wash solutions", also referred to as "wash solvents", are
solutions that are used to wash a vessel or a vessel-to-vessel
connector after it has been vacated by a reagent.
Overview of the Invention in Relation to the Figures
The following, by relating the invention to the Figures, is
intended to exemplify the invention, not limit it.
FIG. 1A shows a computerized system 100 for regulating complex
reaction sequences in small volumes of solution. The system
contains a reaction vessel 71 for holding a volume of fluid 81 and
reagent vessels 1-6 (for holding fluid reagents 11-16,
respectively)., The vessels 1-6 are each connected to a gas source,
selected from a group 171. Gas sources 171a-f each contain a gas
174a-f under pressure. The system also has a rotary valve
combination 59 connected to receive fluid from reagent vessels 1-2
and to deliver fluid to the reaction vessel 71. The combination can
be seen to have other receiving-delivery capabilities. The rotary
valve combination 59 comprises a multi-position rotary selector
valve 51 connected via a conduit means 121 to a rotary switching
valve 61. A computer 17 is connected to the valve combination 59
(Computer connections are described in the text herein but are not
shown in the Figures.) The computer controls both whether the
switching valve 61 is configured to allow or to not allow fluid
flow to the reaction vessel 71 and whether the selector valve 51 is
configured for input from reagent vessel 1 or from reagent vessel
2.
The system is adapted for polymer sequence analysis. To that
purpose it includes a conversion vessel 101 for holding a volume of
fluid 111. It also includes a restraining means 191 for restraining
the unprocessed portion of the polymer in the reaction vessel 71
(See FIG. 7). The restraining means can, as in the example below,
provide a surface to which the polymer can noncovalently adsorb, or
it can be a component to which the polymer, e.g., via an end group,
can be covalently linked. The system also has a rotary switching
valve 62, under the control of computer 17, for controlling fluid
flow to the conversion vessel 101 from the reaction vessel 71. The
system also has a second rotary valve combination 58 connected to
receive fluid from reagent vessels 4-5 and to deliver fluid to flow
to said valve 62. The combination can be seen to have other
receiving-delivery capabilities. The rotary valve combination 58
comprises a multi-position rotary selector valve 52 connected to a
rotary switching valve 65. The computer 17 is connected via a
conduit means 121 to valve combination 58 so that said computer
controls both whether the switching valve 65 is configured to allow
or to not allow fluid flow and whether the selector valve 52 is
configured for input from reagent vessel 4 or from reagent vessel
5.
The system has additional components. It has wash solution vessels
41-44 (for holding wash solutions 141-144), the vessels connected
to a gas sourceselected from group 171. Vessels 41-43 are connected
as optional input to valve combination 59. Vessel 44 is optional
input to valve combination 58. Additionally, the system has an
analytical device 151, an HPLC Column in combination with a UV
monitoring device. Other features of interest are a switching valve
64 under the control of computer 17, for controlling fluid flow to
the HPLC column from the conversion flask 101. Reagent vessels 221
and 222 are sources of reagents 181 and 182 for the HPLC column.
Waste vessels201 collects waste fluid.
Multiposition rotary selector valve 51 is schematically shown in
FIG. 5 and, in the first unit 249 of the valve, comprises a central
common port 241 and a circular array of a plurality of peripheral
ports 231a-j, (only 231a and 231f are marked in FIG. 5) such that
the main axis (imaginary line 135) of the central common port is
the same as the main axis through the circular array. The valve
also comprises, on a second valve unit 245, a radial connector
channel 251 in a second unit 245 of the valve. A continuous
selector channel is created by the common port 241 plus the conduit
means 251 plus the peripheral port 231a. The common port 241 can be
commented to any peripheral 231a-j port selected by rotation of the
second unit as determined by the computer 17.
Rotary switching valve 61 is schematically shown in FIG. 6. The
valve comprises, in a first valve unit 283, a circular array of
four peripheral ports 271a-271d and, in a second valve unit 246,
further comprises connector channels 281 and 282 In FIG. 6, a
continuous switching channel is created by peripheral port 271b
plus connector channel 281 plus peripheral port 271a. A continuous
switching channel is also created by peripheral port 271c, plus
connector channel 282, plus peripheral port 271d. The second unit
246 is rotatable about its main axis (imaginary line 136) under the
control of the computer 17, so that connector channel 281 can form
a continuous switching channel with, peripheral ports 271a and
271d. In the latter situation, channel 282 forms a continuous
switching channel with peripheral ports 271b and 271c.
A rotary valve combination 59 comprising a rotary selector valve 51
connected by its common central port 241 via a conduit means 121
(e.g., a tube or capillary with an internal passage 122) to the
peripheral port 271c of a rotary switching valve 61 is shown
schematically in FIG. 11.
EXAMPLE
Materials
Here, we report development of a novel instrument with miniaturized
reaction cartridge and conversion flask (each about 1/10th the size
of a high-end commercial instrument--Applied Biosystem's model
494cLC) and reagent/solvent flow paths. Our sequencer allows
identification of amino acid derivatives well below 50 femtomoles,
with repetitive yields in the 90-94% range; chemical background is
very low. Because the instrument incorporates a miniature,
pressurized flask with glass capillary pick-up line, it could
conceivably be interfaced with continuous-flow, nanospray-type mass
spectrometry, as recently reported,.sup.31 for mass analysis of any
peptide or amino acid derivatives. The position of the mass
spectrometer would be the same as the HPLC column in FIG. 1.
All reagents and solvents (see also FIG. 1), PTH-amino acid
standard, .beta.-lactoglobulin standard, polybrene (`biobrene`),
glass fibre discs, and cartridge seals were obtained from Applied
Biosystems (Foster City, Calif.), except where noted. Acetonitrile
was from Burdick & Jackson (Muskegon, Mich.), PTH-norleucine
from Pierce (Rockford, Ill.), and the highest purity argon and
helium gas from TechAir (White Plains, N.Y.).
Microfluidics Sytem: Edman Chemistry Configuration
The automated micro-fluidics system 100 (also referred to as the
"PSMSK sequencer" or simply "PSMSK") can be better understood in
relation to FIG. 1. The system is built around an array of eight
rotary valves, connected with fused silica capillary (50, 75, 100,
and 150-.mu.m I.D./365-.mu.m O.D.; Polymicro Technologies; Phoenix,
Ariz.) to two miniaturized reactors, a reaction cartridge
(functioning as a reaction vessel) 71 and a conversion flask (or
conversion vessel) 101 (see FIG. 1). The system has an extremely
low internal volume of all components in the fluid path (see Table
1), which makes possible the automated precision metering and
delivery of reagents in the sub-microliter range.
Cheminert series (Valco, Houston, Tex.) rotary valves were chosen
for the fluid system because of their low internal volumes, small
size, and ease of computerized control. These valves have 150-.mu.m
port diameters and a total internal volume of less than 0.4 .mu.L.
By mounting the valve rotor seal with a lower torque, greater
longevity is obtained at a sealing pressure of 250 psi (instead of
a normal factory-rated 5,000 psi). The wetted surfaces are
fabricated out of the inert polymers, PAEK and Valcon E. PEEK nuts
and one-piece fused silica adapter ferrules (Valco #ZN1FPK-10,
ZF1PK) are used to connect fused silica capillary to the valve
ports. Two basic categories of rotary valves are used in the
system. The multi-position selector valve is characterized by a
central common port and a circular array of peripheral ports
positioned at the outer circumference of the valve stator. A rotor
seal 245 with a single radial fluid path connects the central
common port to one of the peripheral ports, determined by the angle
of rotation. Ten-port port valves 52 and 51 of this type (FIG. 5)
are used as a conversion selector valve 52 and a reaction selector
valve 51 respectively to select reagents and argon gas for delivery
to the conversion flask and reaction cartridge, respectively (FIG.
1). Two-position switching rotary valves have ports positioned
around the outer circumference of the stator only (FIG. 6) The
rotor seal 246 in a switching valve typically has more than one
notch and connects adjacent ports in an arc shaped path. Six-port
switching valves 64 and 63 are used as an injector valve 64 and a
flask valve 63. Four-port switching valves 61, 62, 65, 66 are used
as transfer valve 62, conversion switching valve 65, TFA isolation
valve 66, and reaction switching valve 61 (See FIG. 6). To create
the specialized flow paths for Edman chemical sequencing and to
eliminate any dead volumes in the system, many of the switching
valves incorporate rotors (rotor seals) that are custom fabricated
as to their number of fluid paths. (Valco, Houston, Tex.). Valves
65, 66 and 61 have rotors with only one fluid path instead of the
standard two; the rotor of valve 63 has two fluid paths instead of
the standard three. Ports that are unused are plugged with PEEK
nuts and solid Teflon tubing (Zeus, Orangeburg, S.C.).
By connecting the common port of a selector valve via a capillary
to a peripheral port of a switching valve, the connecting capillary
may serve as a precision metering loop. (FIG. 11) This arrangement
is used to deliver consistent volumes of reagents to both the
reaction cartridge and the conversion flask 101. For instance, the
reaction selector valve 51 is coupled to the reaction valve 61 with
a section of capillary that forms a 0.5-.mu.L loop 121, used to
meter liquid TFA and PITC to the reaction cartridge 71 (FIGS. 1 and
11). The conversion selector valve 52 is coupled to the conversion
switching valve 65 with a section of teflon tubing forming a
7.5-.mu.L loop 124, used to deliver a metered volume of PTH-amino
acid standard, 25% TFA, and 10% MeCN to the flask 101 (FIG. 1). It
is especially useful for the consistent injection of sample to the
HPLC system. The use of this metering loop is described further
below.
Each valve has its own integral microelectric actuator for precise
control of rotation with a 100-msec switching time (actuator not
shown in the Figures). The instrument control software sends simple
ASCII commands via standard RS-232 serial communication to control
the actuator position. Each valve is programmed with its own unique
identity number. The commands are prefaced with this I.D. tag,
allowing the control of several valves simultaneously over one
daisy-chained serial connection. Since the valve rotation is
bi-directional, the valves are initialized by the software to take
the shortest possible rotation path when switching between ports.
This feature is considered strategically when programming valve
functions to ensure that certain chemicals (i.e. TFA and TMA) never
mix (see FIG. 2).
The current system includes twelve 40-mL bottles 1-6, 41-44, 181-2
for a wide range of customized chemistries. Each bottle is
pressurized with argon gas through an inert teflon solenoid valve
(Angar Scientific; Cedar Knolls, N.J.) controlled by logic signals
from the instrument control software of a computer 17. Inert PEEK
check valves (Lee Company, Westbrook, Conn.) are fitted on the
bottle pressure lines to ensure that chemicals do not contaminate
the argon regulation system.
There are six argon regulators 171a-171f that are in gas sources
and which provide argon at pressures of 2.0, 2.0, 4.0, 12.0, 24.0
and 50.0 psi, respectively. (Porter Company, Hatfield, Pa.) (see
FIG. 1). Two are dedicated to pressurizing the TFA bottle 3 and TMA
bottle 2; the remaining four are available for supplying the other
bottle positions, for providing varying levels of pressurized argon
to deliver reagents to the reactors, and for drying operations. The
pressure of each regulator is monitored by a chip-based pressure
transducer (SenSym, Milpitas, Calif.) and displayed in the
instrument status window on the computer.
Reaction Cartridge
There are two reactors in the system when configured for Edman
chemistry, a flow-through `reaction cartridge` 71 and a solution
chemistry `conversion flask` 101 (FIG. 1). Various features of the
reaction cartridge are shown schematically in FIGS. 7, 8 and 9. The
reaction cartridge 71 consists of two 0.5-inch diameter/0.5-inch
high cylindrical blocks 233 (FIG. 7) and 234 (FIG. 8), manufactured
out of borosilicate glass (United Lens; Southbridge, Mass.),
stacked vertically to form the reaction chamber. Both blocks were
bored with a 0.015-inch fluid path 225 through the central axis.
One flat face 226, 227 of each block was machined with a 0.25-inch
cylindrical cavity 237, 238 for a pressure fit tubing connection
utilizing a ferrule. The second flat face 224 of the bottom block
234 was machined with a conical cavity 226 contiguous with a 3-mm
circular cavity 236 to hold a glass fiber support membrane 191 for
immobilizing the protein (FIG. 7). The second face 228 of the top
block 233 was machined with a complimentary mating surface
including a rim 239 that seals precisely around the circumference
of the glass fiber disc with the use of a permeable Teflon seal 235
(See FIG. 9). The second face 228 was also machined to include a
conical cavity 242.
The cartridge 71 is assembled in an aluminum heater (not shown)
that is thermostatically regulated by the instrument control
software. The heater has a window and neon back lighting to view
the contents of the cartridge. The top block 233 is held captive
inside the reaction cartridge assembly at all times. It is only
removed for occasional cleaning. For sample loading, the bottom
cartridge block 234 is removed from the assembly in a stainless
steel holder with a simple "half-twist" mechanism. Once the protein
195 has been pipetted on the disc 191, a permeable teflon seal 235
is placed on top of the bottom block 234 (See FIG. 9). The
cartridge 71 is then fully assembled by mounting the stainless
steel holder (not shown) to the heater with a second half-twist. A
spring steel bellville washer is located at the top of the
cartridge to ensure a tight seal while preventing the glass blocks
from damage during the assembly. The cartridge is pressure tested
prior to all sequencing runs to ensure that the reaction chamber is
an isolated, oxygen-free environment, minimizing background
artifacts produced during the coupling and cleavage reactions.
There are two rotary valves 61 and 62 that control flow through the
reaction cartridge. Located at the bottom of the cartridge is the
reaction switching valve 61, that controls delivery of reagents and
argon gas from the reaction stream selector valve. The direction of
flow during the sequencing chemistry is from the bottom upward
through the sample carrier. The transfer valve 62 at the top of the
reaction cartridge is a two position switching valve, that directs
the outlet of the cartridge to the conversion flask: for further
processing or to the waste bottle.
Conversion Flask
The conversion flask 101, illustrated schematically in FIG. 10, is
a, 96-.mu.borosilicate tapered glass vial (Pierce; Rockport, Ill.).
It is mounted inside an aluminum heater (not shown)
thermostatically regulated under the control of the computer 17.
Tubing connections are made to the flask through a teflon seal 102
at the top. The vial can be removed easily from the heater for
cleaning or exchange by unthreading a stainless steel thumbscrew
located at the bottom of the assembly. There are two tubing
connections 131, 132 to the flask 101. The first is a vent line 131
made from 1/16-inch O.D./0.004-inch I.D. teflon tubing. It is
installed with a simple pull-through friction fit through a
0.056-inch hole in the teflon seal. The vent line is cut flush with
the inside of the teflon seal using a razor blade. The second
tubing connection to the flask is a pickup line 132 made out of
75-.mu.m I.D./365-.mu.m O.D. Polyimide coated fused silica
capillary. To install the pickup line first a 0.01-inch
I.D./0.0625-inch O.D. teflon tubing sleeve 137 is pulled through a
0.056-inch hole in the teflon seal at the top. This sleeve is cut
off flush with the inside of the teflon seal using a razor blade.
By pulling the capillary 132 through the teflon sleeve a pressure
tight seal is formed. A long enough section of capillary is pulled
through the teflon seal so that it reaches the bottom of the flask.
A bunsen burner is used to burn off the polyimide coating from the
portion of the pickup line that is exposed to reagents, as it was
found that the polyimide breaks down during the conversion process
and interferes with the liquid chromatography.
A 6-position, 2-port switching valve 63 (see FIG. 1) is located
above the flask 101 and controls the delivery of fluids and argon
gas. In position A, the vent is opened to waste and the pickup line
132 is connected to the transfer valve 62. This position is used to
deliver fluids to the flask 101 in a controlled fashion from the
bottom up, and also to bubble argon through the sample. This argon
dry operation is used in many situations, including the conversion
of ATZ-amino acids to their PTH counterparts, solvent evaporation
prior to HPLC injection, and high pressure flask cleaning between
cycles. In position B, the vent line 131 is connected to the
transfer valve 62 and the pickup line 132 is connected to the
injector valve 64. This position is used to empty the contents of
the flask 101 to the injector loop 123 or the waste bottle 201. By
delivering pressurized argon from the reaction selector valve 51 to
the vent line 131 the contents of the flask 101 are forced out
through the pickup line 132.
A 10-cm long, 50-.mu.m I.D. restrictor capillary 134 is connected
to the outlet of the injector valve 64. The narrow internal
diameter of the restrictor capillary provides sufficient back
pressure to maintain a gradual 15 second loading of the injector
loop 123 when argon at 12.0 psi from gas source 171d is used to
pressurize the flask. The low internal volume of the restrictor
capillary is also conducive to a high injection percentage. When
the first sign of liquid is present at the end of the restrictor
capillary, only 0.2-.mu.L of sample has passed beyond the loop.
This makes it possible to routinely inject 5 .mu.L out of a sample
volume of 7.5 .mu.L.
HPLC System
PTH-amino acids are separated on a 25-cm long, 300-.mu.m I.D.
capillary HPLC column, 151 custom packed (LC Packings, San
Francisco, Calif.) with 5-.mu.m C.sub.18 PTH stationary phase. The
column 151 is placed inside a heated enclosure (Eppendorf;
Westbury, N.Y.)) and thermostatically regulated at 55.degree. C.
HPLC solvent A 221 is 5% tetrahydrofuran in water, with 20 mL of
`Premix Buffer` added per liter (Applied Biosystems); solvent B 222
is acetonitrile (Burdick & Jackson) with 500 nmol of
dimethylphenylthiourea (DMPTU; Applied Biosystems) added per liter.
Solvents are contained in Kontes Ultraware (Vineland, N.J.)
reservoirs 181 and 182 and are sparged with helium gas at a
pressure of 3 psi. The gradient is formed by an Applied Biosystems
140D micropump at a flow rate of 30 .mu.L/min; a flow splitter (LC
Packings) with a 10:1 split ratio delivers a consistent gradient
(12-24% B/4 min; 24-50% B/22 min; 50-90% B/1 min) to the column at
a flow rate of 3 .mu.L/min. The flow splitter 232 also contains a
micro-volume union and static mixer. The outlet of the column is
directly connected to a UZ-View `ball lens` capillary flow cell (LC
Packings) which is mounted in an Applied Biosystems model 785A UV
absorbance detector. All connections in the LC system are made with
75-.mu.m I.D./365-.mu.m O.D. fused silica capillary. The detector
is set to a wavelength of 269 nm, with a rise time filter setting
of 2 sec. The absorbance data is collected from the UV detector by
a Nelson 900 Series analog to digital converter, filtered, stored,
and analyzed using TurboChrom 4 software (PE Nelson; Cupertino,
Calif.).
Instrument Control
The instrument 100 is controlled by a computer 17, a Macintosh 225
MHz computer (Apple; Cupertino, Calif.) comprising and running a
software application, which was programmed in the LabView
environment, version 5.1 (National Instruments, Houston, Tex.). A
DaqCard 1200 (National Instruments) data acquisition and control
card is installed in the computer and is used to interface between
the software and circuit board contained in the instrument
enclosure. The DAQ card has 8 analog to digital (A-D) converters
and 24 lines of digital input/output. The A-D converters monitor
the signals from the reaction cartridge and conversion flask
temperature sensors as well as the argon regulator pressure
transducers. The digital lines control the solenoid valves,
reaction cartridge and conversion flask heater elements, and four
relays used to control external devices (e.g. HPLC pump, UV
detector, and data collection system). The rotary valves are
controlled by serial communication directly from the computer's
serial port. Communication from the computer to the sequencer is
accomplished through one DB-9 RS-232 serial cable and one 50
conductor ribbon cable. These connect to ports on the back panel of
the instrument. Also located on the back panel are a cooling fan,
bulkhead connection for the waste bottle vent line, and the four
relay outputs for controlling external devices.
The circuit board is multifunctional. It buffers the low millivolt
signals from the pressure transducers and temperature sensors and
converts them to the useable range of the DAQ card A-D converter.
The 5 VDC digital logic signals from the DAQ card trigger
transistors on the circuit board to switch higher current power to
the solenoid valves, relays, and heater elements. In the case of
the solenoid drivers, an additional circuit drops the initial 24
VDC `strike` voltage down to 12 VDC after 100 msec. The board also
acts as a voltage regulator, supplying the correct power to the
instrument's cooling fan and neon lighting. The circuit board was
created from a copper clad board with a `photo-resist"` coating
(Kepro Circuit Systems, St. Louis, Mo.). A negative of conductor
circuitry, designed with CAD software, was printed on a
transparency, and placed on the board, after which the board was
exposed to UV light in a darkroom. Hot sulfuric acid was used to
etch away unneeeded copper, leaving the desired conductor
circuitry.
The Lab View software environment allowed the design of a graphical
display that emulated the actual operation of the instrument. The
status of all valves, relays, regulator pressures, and temperature
transducers are displayed on the monitor screen 18 in real time as
the instrument is operated. The software has two modes of
operation, manual and automatic. In both modes the same real time
graphical display is utilized. In the manual mode a manual control
window is also present on the computer desktop. This window
contains buttons for the control of every piece of hardware at the
user's discretion. In the automatic mode, control cycles are
preprogrammed by the user. In this case, the manual control window
is replaced by a sequencer status window that displays information
regarding the current sequence, cycle, and function being
performed.
An automated control program is created through the use of
user-defined reaction and conversion functions (see Table 2). A
sequencer cycle is one complete program for analyzing one amino
acid residue of the protein. Distinct reaction and conversion
cycles are created independently to control the activity of the two
distinct reactors 71, 101. The software then meshes these two
cycles together automatically so that the transfer of sample
between the reaction cartridge 71 and conversion flask 101 is
coordinated properly. Several cycles are looped together to create
a sequence program that can process as many amino acid residues as
the user requires. Sequencer functions are not necessarily single
command events. A function can include as many independent timed
`events` as needed. In fact, nearly all of the functions we used
were more than one event, with a maximum of eight events. This
feature is especially useful since most of the chemistry is
automated with the use of metering loops. Delivering a loop of
reagent is a multi-step procedure, as illustrated by the metered
delivery of TFA liquid 13, the most complex case, in FIG. 2.
Operation
The reaction cartridge 71 was designed with a 3-mm diameter disc
191 to reduce chemical and amino acid background, a requirement to
call amino acid sequence from the chromatographic data. Yet, the
size should be large enough to allow for a practical sample loading
volume. At a diameter of 3 mm, a glass fiber disc can hold a sample
volume of 2 .mu.L. Several 2-.mu.L loads to a single filter can be
done in series to accommodate samples of larger volume. Prior to
sample loading, the glass fiber disc 191 is coated with 0.18 mg of
polybrene, a polymeric sample carrier that increases the protein
affinity for the glass fiber. The unloaded, polybrened disc is then
precycled by running several standard sequencing cycles until the
chromatogram appears clean. BLG protein standard was pipetted onto
the disc, dried using 2-psi argon gas, and the reaction cartridge
was reassembled and pressure tested. The reaction and conversion
cycles (Table 2) were initially based on the custom cycles used
with commercially available sequencers in our lab..sup.17 They have
since been extensively modified to meet the requirements of the new
miniaturized flow path and adjusted according to experimental
observations.
Table 2A illustrates in detail reaction cycle #27 "Glass Fiber
Disc". Steps 1-II comprise the coupling of PITC 11 to the
N-terminal amino acid of the protein. TMA vapor 12a originating at
2 psi from solution 12 is delivered three distinct times, at 400
seconds each, for a total of 1200 seconds. TMA provides the basic
environment necessary for proper coupling. Deliveries of 1-.mu.L
aliquots of 1% PITC 11 are interspersed between the TMA steps. The
overall coupling time in the reaction cycle was set intentionally
long to ensure minimal sequencing lag that would otherwise result
in inefficient coupling chemistry. This is then followed by a
series of solvent washes (Steps 12-29) with heptane 141, ethyl
acetate 142, and butyl chloride 143. The volume of the washes was
optimized to minimize chemical background while preventing sample
washout (see Table 1). For subsequent acid cleavage (Step 30),
delivery of TFA 13 was also tightly controlled, as to achieve low
lag while minimizing sample washout. We found that a volume of 0.5
.mu.L was most effective. This step is described in detail in FIG.
2. Transfer of ATZ-amino acids to the conversion flask 101 (Steps
36-55) was the subject of rigorous optimization. Since the volume
of the flask is only 75 .mu.L, solvent volume should balance
efficient extraction of the ATZ-amino acids with this limitation.
Thus, the delivery of the transfer solvent (BuCl 143) was broken up
into several smaller steps, with argon dries interspersed between,
to keep the solvent in the flask at a manageable level. This
procedure also increases the efficiency of the extraction, as
shown..sup.17 Following the transfer phase, the reaction valves are
cleaned with BuCl, 143, Heptane 141, and EtAc 142, to prepare them
for the next amino acid cycle (Steps 58-61).
Table 2B details the conversion cycle utilized, #28 "Low Pressure
Conversion". It begins with an extensive cleaning of the flask 101
(Steps 1-10) with 25% TFA/H2O 14 and 10% MeCN/H2O 144 to eliminate
any carryover between cycles. The cleaning solvents are delivered
through the vent line 131, instead of through the pickup line 132
as is the case with other deliveries during the sequencing run,
ensuring better cleaning of all fluid pathways. Once filled with 40
.mu.L solvent, the highest available argon pressure (50 psi) from
gas source 171f is bubbled through the pickup line 111+132/lower
section to vigorously wash the entire internal volume of the
flasks, including the top teflon seal and the highest reaches of
the vial. The flask valve 63 then switches position so that high
pressure argon is delivered to the vent 131, forcing the contents
of the flask 101 to the injector waste 201. To expedite the large
volume deliveries of 25% TFA/water 14 and 10% MeCN/water 144
through the vent line, these bottles are pressurized with argon at
12.0 psi from source 171d. After cleaning, a 225-femtomole,
7.5-.mu.L aliquot of PTH-norleucine 16 is added to the flask and
evaporated (Steps 11-12). Then, a 7.5-.mu.L loop load of the
conversion reagent, 25% TFA/water 14, is delivered to the flask 101
(Steps 13-14), just before the transfer of ATZ-amino acid from the
reaction cartridge 71 (Step 15). Argon at 12.0 psi is bubbled
through the flask 101 for better conversion reaction kinetics and
to slowly evaporate off the 25% TFA (Step 16). Once the flask 101
is completely dry, the injector loop 123 is flushed with argon to
prepare for injection (Step 17), and the contents of the flask 101
reconstituted with a 7.5-.mu.L loop of 10% MeCN, and bubbled with
argon from source 171e at 24.0 psi to improve dissolution (Steps
18-22). Argon at 12.0 psi is then delivered to the vent line 131,
displacing the sample into the injector loop 123 (Step 23). After
15 seconds, the injector valve 64 switches position, injecting the
sample onto the column 151 and a relay closes to start the HPLC
gradient program (Steps 24-25).
RESULTS AND DISCUSSION
Proteins can be prepared, fractionated and detected at the
femtomole level. Taking into account a 2 to 5-fold loss during
sample handling, chemical structure determination must then be done
with just 100-200 femtomoles of starting material. Our objective,
therefore, was development of an automated protein sequencer whose
range of operation would include that range. We sought to do so by
further miniaturizing the entire fluidics system, including
reaction vessels and the analytical component, capillary
(300-micron ID) LC, all in a very proximate arrangement.
Preliminary Considerations
We first constructed a stand alone, modular capillary-LC system
(see Materials and Methods). Flow rates, gradients and column
temperatures were optimized for various column/solvent combinations
as to yield the best possible resolution, highest signal/noise
ratio (in general and for each PTH-aa in specific) and least
baseline drift. In anticipation of `on-line` sample injections from
a sequencer, we studied tolerance for increasing MeCN
concentrations (3 to 10%) in function of progressively larger
injection volumes (0.5 to 20 .mu.L). As `sequencer-injected`
samples may also contain trace amounts of TFA, its effects were
also investigated. Initial attempts at optimizing operational
parameters, injecting standards (20-100 femtomoles each of all
PTH-aa) in an 0.5-.mu.L volume of 3% MeCN/0.1% TFA, resulted in
complete separation. Most PTH-aa's can be detected at the 20
femtomole level, with signal/noise ratios better than 1 to 5, and
injections of up to 5-8 .mu.L in volume appeared feasible (data not
shown). Consequently, if analysis of 60-70% or more of the PTH-aa's
after each cycle is desired, the analytes should be dissolved in
less than 7-10 .mu.L of solvent, in turn necessitating a smaller
conversion flask; e.g. 75 .mu.L, as compared to 750 .mu.L in
current commercial sequencers. Space constraints required the use
of 75-.mu.m I.D./365-.mu.m O.D. capillaries for liquid delivery and
pick-up. To avoid overfilling the smaller flask 71 during ATZ-aa
transfer, the reaction disc 191 had to be reduced in size
accordingly, to 3-mm diameter. Further reduction in wetted surfaces
was accomplished by bringing chemistry vessels and detection system
in close proximity, plumbed together with 50 to 100-micron ID
capillary tubing as well, to create an integrated micro-fluidics
system. Capillary bore tubing requires substantially higher
pressures for delivering and drying liquids. Using a bread board
instrument, it was found that increased back pressures cause
liquids to back up in the wrong ends of the Z-path type valve
blocks of an Applied Biosystems instrument. It was therefore
decided to replace them with nanoliter-dead volume rotary valves in
the final design, which also permitted accurate metering of
reagents in the sub-microliter range.
Design and Construction
The instrument was constructed to conform to the stipulations
summarized above and following the schematic diagram shown in FIG.
1. It features the incorporation of 8 rotary valves (numbered in
FIG. 1). The reaction cartridge 71 is positioned between valves 61
and 62 (liquid and gas flow from the bottom up) and the flask 101
right below valve 63. Note that, together, valves 51 (10-port
selection) plus 61 (two-way switching) accomplish the same result
as the "cartridge reagent/solvent-blocks" in an Applied Biosystems
automated sequencer (see also FIG. 2); similarly, valves 65 and 52
accomplish the same result as the "flask reagent-block" in an
Applied Biosystems automated sequencer. Valve 66 (FIGS. 1, 3)
serves to isolate TFA from the rest of the chemicals and solvents
to exclude potential chemical problems, including salt formation
upon contact with base (TMA). Rotary valves were selected on the
basis of the lowest-dead volume and compatibility with TFA. Acid
was continuously pumped through for an entire week without evidence
of pressure leaks or visual damage. Cartridge 71, flask 101 and
LC-injector valve 64 plus column 151 are all plumbed with glass
capillary tubing; the rest with teflon capillary tubing.
The level of miniaturization and versatility incorporated in the
design of our automated micro-fluidics system, as described here in
the PSMSK sequencer, is best illustrated by comparison of the
chemistry modules, selected wetted surfaces and variable
pressurization to those in a state-of-the-art, commercially
available sequencer (Applied Biosystems model "cLC494"). As shown
in Table 1, the PSMSK reaction disc/chamber is about 4-8 times
smaller, and eightfold less reagent and considerably less solvent
are consumed; however, wash solvent per disc surface area is
comparable. The overall flow path volume(E-block to injector) is
30-fold smaller as compared to the cLC494. Available gas pressures
range between 2 and 50 psi, whereas only from 1 to 3.5 psi in a
commercial instrument. Most importantly, only the current system
permits reproducible injection after every chemistry cycle of 67%
of the end-products in a 5-.mu.L volume, as compared to 55% in
satisfying a critical requirement for the practical use of any
300-micron ID LC-columns.
Automation control software was written using Labview software on a
Macintosh platform (details on software design package, digital
control interface and computer are given in `Materials and
Methods`), and sequencer cycles (about 50-70 min in length; see
Table 2) developed, allowing fully automated operation of the
instrument 100. In addition to required temperatures, pressure
regulation and gas flows, the program controls all rotary plus
solenoid valves simultaneously and in a fully coordinated fashion;
and allows to string together an essentially unlimited number of
"functions" to create cycles of chemistry ("cycles"); which, in
turn, can be looped together (i.e. repeated) 20 or more times. Any
function itself consists of a particular sequence of unique valve
position combinations ("events"), as illustrated in FIG. 2 for
function #29, `deliver liquid TFA to cartridge`, a seven-event
function. Here, sequentially, the reaction valves are flushed with
argon, TFA metered and delivered to the cartridge, valves flushed,
reaction selector valve rinsed with EtAc, flushed with argon and
valves turned into `safe` position (i.e. central channel in line
with plugged port). Operational status can be monitored at all
times through the use of a `virtual instrument` graphical display
that emulates actual instrument operation.
Operation and Fine Tuning
The prototype instrument 100 was fully tested for automated
operation, followed by optimization of flows and chemistries, and
then interfaced with a capillary HPLC system. To this end, precise
optimizations of ATZ-extraction/delivery to flask and PTH-aa
transfer from flask 101 to injector valve 64 were carried out,
largely empirically, with the objective of reducing extraction and
transfer volumes. Additional issues addressed were conversion
chemistry (conditions of initial aqueous ATZ to PTC conversion, and
TFA concentration for generating PTH-aa's), and dissolution
(volume; % MeCN/% TFA) and volume reduction (forced argon
evaporation) of the PTH-aa's. As a result, the system enables near
quantitative LC-injection of volumes in the 5-10 .mu.L range,
making it fully compatible with capillary-LC.
This is most convincingly illustrated in FIG. 3, where an LC
separation of 25 femtomoles of PTH-amino acid standard 15 is shown.
Note, that it concerns here 25 femtomoles of standard delivered
into the conversion flask, dried down, redissolved, volume reduced
by forced evaporation, and injected for analysis. We measured the
final volume in the flask to be about 7-8 .mu.L. Of that final
volume, 5 .mu.L (67%) is then normally injected, representing 17
femtomoles of standard mixture, assuming that no losses have
occurred in the process.
Protein Sequencing
Following optimization of the individual chemistry and analysis
modules, the integrated system was tested for micro-sequencing
performance using, first, 1-pmole and then 400-fmole amounts of
non-covalently attached protein standards. Changes were introduced
to redress any observed problems until initial yields (i.e.
recovery of the first amino acid) were consistently over 50%,
repetitive yields (i.e. efficiency of the Edman degradation
chemistry) in excess of 90%, and the predominant chemical
background (i.e. DPTU) belong 1 piciomole per cycle. Several
thousand cycles have since been completed without any major
problems.
An example of the PSMSK cyclic sequencing chemistry, using 100
fmoles of beta-lactoglobulin as starting material, is shown in FIG.
4. Although we recognize that the lower limits of sensitivity are
being approached in this instance, the sequence can actually be
called quite well by those skilled in the art, with the exception
perhaps for Thr and Lys, residues known to recover at substantially
lower yields during Edman degradation..sup.17 The initial yield was
well over 70%, even after background subtraction; repetitive yield,
as calculated from the background subtracted yields of PTH-Val in
cycles 3 (74 fmoles) and 15 (21 fmoles), was 90.0%. By all means,
this would be considered an acceptable result for any sequencing
experiment in the event when a system's, any system's, lower limits
of sensitivity have been reached. The difference being that in the
example presented here, the amount of starting material was about
eight to ten times less than hitherto reported for some of the more
challenging high-sensitivity experiments using state-of-the-art
commercial instruments..sup.19,23,24 Our result exceeds
manufacturer performance specifications (i.e. 15 residues of 2
pmoles beta-lactoglobulin, with 92% repetitive yield).sup.19 of an
Applied Biosystems cLC494 instrument by at least an order of
magnitude.
The PSMSK instrument represents, therefore, a new phase in
operative, Edman-chemical protein sequencing, both in terms of
miniaturization and sensitivity. To put this advancement in some
historical context, a brief overview of the Edman chemistry and its
automation during the second half of the 20th century is given in
Table 3.
Practical Considerations
To function as a truly practical and reliable device for elaborate,
constantly repeated cycles of chemistries and analyses, be it
process- or micro-scale, certain standard criteria of versatility
and routine operation needed to be satisfied. Among PSMSK's most
desirable operational features, we will briefly consider its
durability and its capacity to utilize large numbers of
reagents/solvents in an essentially unlimited sequence of
reactions, extractions, drying steps, dissolutions, and
transfers.
In the application-validated protein sequencer embodiment described
above, ten different `liquids` 11-16, 141-144 were transferred
through diverse combinations of flow-paths and vessels, by using
changing gas pressures and value position combinations, to carry
out 86 discrete steps (61 in the reaction cycle; 25 in the
conversion cycle--Table 1), totaling 227 `events` per cycle. While
permitting a large degree of flexibility already, our automated
microfluidics system could easily be upgraded to deliver dozens
more of chemicals, through increasingly elaborate flow paths, by
adding a few more multi-port selection valves. Such additions would
be readily supported by the Labview-based instrument control
software.
In cases of frequent usage, it is conceivable that dozens of
`cycles`, hundreds of `steps` and several thousand `events` would
be completed per day, totaling tens of thousands of valve rotations
per week, and raising legitimate concerns about abrasion of rotary
seals and resultant blockage or leakage. However, this is not what
we have observed after more than a year of regular usage,
comprising hundreds of thousands of discrete valve movements. Aside
from offering some of the lowest internal dead volumes available,
`Valco Cheminert` microbore-type rotary valves (see `Methods and
Materials`) have originally been designed to withstand pressures of
up to 3,500 psi and could therefore be slightly loosened (e.g.
factory calibrated to 200 psi) to accommodate a maximum pressure of
50 psi in our instrument, thereby extending life expectancies.
Finally, since the instrument is plumbed with capillary tubing, we
took extra precautions to prevent salt crystal formation by
separating acid and base in the microfluidics system at all times.
This was most easily accomplished by isolating TFA 13 on a
dedicated valve 66, in conjunction with proper rinsing of the
exposed flow paths with EtAc 142 and flushing with argon from three
different ports (see FIG. 2). As a result, no particulate-relating
problems have ever been observed.
CONCLUSIONS
Detailed biochemical analysis of all molecular communications in a
cell will require better tools for microchemical quantitation and
identification. We sought to assemble and optimize a
microfluidics-based instrument for automation of serial chemical
and enzymatic reactions on the smallest possible scale, while
maintaining the capacity to analyze a large portion of the
end-products. Here we describe a miniaturized, fully integrated and
automated system, consisting of multiple rotary valves, reaction
and collector modules, all connected by capillary lines, that can
deliver about 70% of the end-products, in a 5-.mu.L volume, to any
analytical device with low flow-injection or -infusion (e.g. cLC or
NanoESI-MS). A near total control of flow path combinations and
directions, temperatures and gas pressures enables precise
execution of complex biochemical laboratory procedures. Instrument
performance was clearly demonstrated by partially sequencing 100
femtomoles of an intact protein using classical Edman chemistry in
combination with capillary-bore liquid chromatographic
identification. To our knowledge, this is the smallest amount of
protein ever reported to be successfully analyzed in this way. Near
quantitative transfer of reaction end-products from a pressurized
micro-vial, in a minimal volume and at exceedingly low flows, could
male any biochemical process fully compatible with NanoESI MS or
MS/MS analysis;.sup.31,32 for instance, protein digestion in
combination with chemical modification(s).
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Table 1. Comparison of automated protein sequencing instruments:
Applied Biosystems `cLC494` and `PSMSK`.
RXN, reaction; CNV, conversion; Evalve, cartridge reagent/solvent
block or valve; other abbreviations, see key in FIG. 1 or Glossary.
R1 corresponds to 11 in FIG. 1C, R3 to 13, R4 to 14, S1 to 144d,
S123 to each of 144a, 144b, and 144c.
"Flask" in the table refers to the conversion vessel 101.
Tables 2A and 2B. Model `PSMSK` reaction and conversion cycles.
RXN, reaction; CNV, conversion; NLE-Std, PTH-norleucine (30
fmol/.mu.L acetonitrile); reaction temperature, 48{character
pullout}C; conversion temperature, 64{character pullout}C. Total
reaction cycle time, .about.68 min; total conversion cycle time,
.about.45 min; further details, see FIG. 1 and elsewhere in the
application.
Table 3. Brief history of Edman protein sequencing.
TABLE 1 A. Wetted Surfaces: RXN Disc RXN Block Diameter Surface
Volume Cavity Volume System mm mm.sup.2 .mu.L .mu.L cLC494 6 28 8
32 PSMSK 3 7 2 4 Flow Path Volume Injector Evalve-Flask
Flask-Injector Total Loop Volume System .mu.L .mu.L .mu.L .mu.L
cLC494 248 130 378 50 PSMSK 11 1.5 12.5 5 B. Reagents/Solvents
Delivery: RXN WASH (S123) Disc Polybrene R1 R3 Volume Vol./Area
System mm mg .mu.L .mu.L .mu.L .mu.L/mm.sup.2 cLC494 6 0.75 8 32
920 33 PSMSK 3 0.18 2 4 270 39 Injec- tion ATZ Transfer CNV
Precent- Volume Vol.Area S4(preATZ) R4 S4(PTH) age System .mu.L
.mu.L/mm.sup.2 .mu.L .mu.L .mu.L % cLC494 291 10.4 60 10 91 55
PSMSK 76 10.9 7.5 7.5 7.5 67
TABLE 2A Reaction Cycle - #27 Glass Fiber Time Elapsed (sec) Step
Description Core Step 1 Deliver TMA to Cartridge 20 68 2 Deliver
PITC to Cartridge 10 35 3 Pause 20 20 4 Deliver TMA to Cartridge
400 448 5 Deliver PITC to Cartridge 10 35 6 Pause 20 20 7 Deliver
TMA to Cartridge 400 448 8 Deliver PITC to Cartridge 10 35 9 Pause
20 20 10 Deliver TMA to Cartridge 400 488 11 AR4 to Cartridge 240
248 12 Deliver Heptane to Cartridge 60 83 13 Pause 10 10 14 AR4 to
Cartridge 10 18 15 Deliver Heptane to Cartridge 60 83 16 Pause 10
10 17 AR4 to Cartridge 10 18 18 Deliver BuCl to Cartridge 60 83 19
Pause 10 10 20 AR4 to Cartridge 10 18 21 Deliver EtAc to Cartridge
60 83 22 Pause 10 10 23 AR4 to Cartridge 10 18 24 Deliver EtAc to
Cartridge 60 83 25 Pause 10 10 26 AR4 to Cartridge 10 18 27 Deliver
EtAc to Cartridge 60 83 28 Pause 10 10 29 AR4 to Cartridge 150 158
30 Deliver Liquid TFA to Cartridge 10 104 31 Load RXN Loop with
EtAc 15 23 32 Clear RXN Loop to Waste with AR5 10 10 33 Load RXN
Loop with EtAc 15 23 34 Clear RXN Loop to Waste with AR5 40 40 35
AR4 to Cartridge 20 248 36 Prep Transfer 10 10 37 Deliver Heptane
to Midpoint 12 32 38 Transfer Pause 15 15 39 Transfer with BuCl
Part 1 15 15 40 Transfer Pause 15 15 41 Transfer with AR4 45 55 42
Transfer Argon Pulse 40 40 43 Transfer with BuCl Part 1 15 15 44
Transfer Pause 10 10 45 Transfer with AR4 30 40 46 Transfer Argon
Pulse 30 30 47 Transfer with BuCl Part 1 15 15 48 Transfer Pause 10
10 49 Transfer with AR4 30 40 50 Transfer Argon Pulse 30 30 51
Transfer with BuCl Part 1 15 15 52 Transfer Pause 10 10 53 Transfer
with AR4 30 40 54 Transfer Argon Pulse 30 30 55 End Transfer 1 1 56
Deliver BuCl to Cartridge 30 53 57 AR4 to Cartridge 120 128 58 Load
RXN loop with BuCl 60 68 59 Load RXN loop with Heptane 60 68 60
Load RXN loop with EtAc 60 68 61 AR4 to Cartridge 120 128
TABLE 2B Conversion Cycle - #28 Low Pressure Conversion Time
Elapsed (sec) Step Description Core Step 1 Empty Flask to Injector
60 68 2 Rinse Vent/Flask with 25% TFA 45 72 3 Rinse Vent/Flask with
25% TFA 45 72 4 Dry Flask with AR6 60 68 5 Empty Flask to Waste
with AR6 400 408 6 Rinse Vent/Flask with 10% MeCN 30 52 7 Rinse
Vent/Flask with 10% MeCN 30 52 8 Dry Flask with AR6 60 68 9 Empty
Flask to Waste with AR6 300 308 10 Dry Flask with AR5 60 68 11 Load
CNV Loop with NLE-Std 10 16 12 Deliver CNV Loop to Flask with AR5
80 80 13 Load CNV Loop with 25% TFA 15 21 14 Deliver CNV Loop to
Flask with AR5 20 20 15 Ready to Receive 1 1 16 Deliver CNV Loop to
Flask with AR4 1000 1000 17 Load Injector Loop with AR5 240 248 18
Load CNV Loop with 10% MeCN 10 16 19 Deliver CNV Loop to Flask with
AR5 20 20 20 Pause 10 10 21 Deliver CNV Loop to Flask with AR5 20
20 22 Pause 10 10 23 Load Injector Loop with AR5 15 23 24 Relay 1
On: Start HPLC 2 2 25 Relay 1 Off 2 2
TABLE 3 Brief History of Edman Protein Sequencing YEAR SCALE
REFERENCES 1950 Edman Chemistry 1 mmol 9,10 1955-56 Practical
Manual 1 mmol 33 Sequencing 1967 Spinning-Cup Sequencer 200 nmol 11
1968-77 Various Improvements 10-100 nmol 13,34 1978 Picomole HPLC
analysis 0.5-5 nmol 14 1981 Gas-Phase Sequencer 20-100 pmol 15 1986
`On-line` HPLC 5-10 pmol 1987-94 Various Optimizations 2-5 pmol
16,17,18 1996 Applied Biosystems cLC 1 pmol 19 2000 Microfluidics
(PSMSK) 100-200 fmol this patent application
* * * * *